Sep 30, 2023

ADP Glo Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr3kqbvmk/v1
  • 1University of California, Berkeley;
  • 2Aligning Sciences Across Parkinson's
Annan SI Cook
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr3kqbvmk/v1
Protocol Citation: Annan Cook, Xuefeng Ren 2023. ADP Glo Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3kqbvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88510
Keywords: ASAPCRN, catalytic activity of the lipid kinase vps34, lipid kinase vps34, glo protocol protocol for adp glo, adp glo, small unilammelar vesicle, glo protocol protocol
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
Protocol for ADP Glo (Promega) assay to measure the catalytic activity of the lipid kinase VPS34 on small unilammelar vesicles.
Attachments
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852-2202.pdf
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Materials
Materials

  • Small unilamellar vesicles (SUVs) composed of
A
40% dioleoyl phosphatidylcholine (DOPC)
20% dioleoyl phosphatidylethanolamine
20% dioleoyl phosphatidylserine
20% liver phosphatidylinositol
All lipids from Avanti Polar Lipids.

  • HEPES pH 7.5
  • NaCl
  • MgCl2
  • MnCl2
  • TCEP
  • ATP
  • PI3KC3-C1
  • ADP-Glo ATP Depletion Reagent (Promega)

Equipment

  • Glass tube
  • N2 stream source
  • Vacuum dessicator
  • Benchtop centrifuge
  • Sonicator with a small probe tip
  • Water bath at 37°C
  • Liquid nitrogen
Preparation of Small Unilamellar Vesicles (SUVs)
15m
Dehydrate 0.4 mg of the lipid mixture (40% DOPC, 20% DOPE, 20% DOPS, 20% liver phosphatidylinositol, Avanti Polar Lipids) in a glass tube using a gentle N2 stream.
Allow the lipids to dry Overnight in a vacuum desiccator to remove excess chloroform.
Rehydrate the dried lipids by adding 25 millimolar (mM) HEPES 7.5 and 200 millimolar (mM) NaCl.
Vortex the mixture to resolubilize the lipids.
Perform 10 cycles of freeze-thaw by immersing the lipids in liquid nitrogen for a few seconds and then in a 37 °C water bath.
Sonicate the lipids using a small probe tip sonicator at 50% power in 2-second on/off cycles for a total of 00:15:00 On ice and a water mixture.
15m
Preparation of Buffers
Prepare a kinase dilution buffer.

Kinase dilution buffer
AB
HEPES pH 7.525 mM
NaCl200 mM
MgCl210 mM
MnCl21 mM
TCEP2 mM

Prepare a 2x lipid dilution buffer.
2x lipid dilution buffer
AB
HEPES pH 7.525 mM
NaCl200 mM
MgCl220 mM
MnCl22 mM
TCEP4 mM

ADP-Glo assay
1h 40m
In a reaction tube, combine the following components:
AB
50 nM PI3KC3-C19 μL
0.1 mg/mL SUVs in the 2x lipid dilution buffer9 μL
500 μM ATP in the same buffer2 μL

Mix the components gently.
Allow the reaction to proceed for 01:00:00 at Room temperature .
1h
After the incubation, add 20 µL of the ADP-Glo ATP depletion reagent to each reaction tube.
Incubate the reaction tubes with the ADP-Glo ATP depletion reagent for 00:40:00 at Room temperature .
40m
Measure the luminescent output for each condition tested using a luminometer or a plate reader.